Patent application title: METHOD FOR INCREASING VIRIDIFLOROL CONTENT IN TISSUES
Inventors:
Ajit Kumar Shasany (Uttar Pradesh, IN)
Pallavi Yadav (Uttar Pradesh, IN)
Shubhra Rastogi (Uttar Pradesh, IN)
Syed Uzma Jalil (Uttar Pradesh, IN)
Rajendra Singh Bhakuni (Uttar Pradesh, IN)
IPC8 Class: AC11B902FI
USPC Class:
1 1
Class name:
Publication date: 2021-12-30
Patent application number: 20210403827
Abstract:
The present invention relates to the development of high viridiflorol
containing variety for enhancing the aroma as well as medicinal
properties of Mentha piperita by overexpressing MPTPS4 gene through
genetic transformation. This transgenic plant possesses better growth and
able to produce essential oil with good quality contains high
viridiflorol maximum of about 25% and proportionate decrease in menthone,
menthofuran and menthol of Mentha piperita as compared to other control
genotype. Mentha piperita (CIM-madhuras) that produces viridiflorol, a
molecule of potential demand in perfumery, cosmetics, toiletries, drugs
and sanitation products.Claims:
1. A method for increasing viridiflorol content in tissues, said method
comprising the steps; a) isolating sesquiterpene synthase gene (MPTPS4)
from Mentha piperita having SEQ ID NO: 4; b) cloning of gene MPTPS4
obtained at step (a) into pHANNIBAL vector to obtain a cassette with SEQ
ID NO: 8; c) re-cloning the cassette obtained at step (b) into pART27
binary vector system with SEQ ID NO: 9; and d) transforming the vector
obtained at step (c) into Agrobacterium tumerfaciens for overexpression
of MPTPS4 and further transfecting into tissues to obtain the increased
viridiflorol content.
2. The method according to claim 1, wherein the terminator sequence is an octapine synthase (OCS) terminator sequence set forth in SEQ ID NO: 10.
3. The method according to claim 1, wherein the promoter sequence is a tissue specific promoter (Limonene synthase, 797 bp) sequence set forth in SEQ ID NO: 7.
4. A transgenic plant obtained by the method according to claim 1.
5. The plant according to claim 4, wherein the plant has increased viridiflorol content up to 25%.
6. A method of increasing aroma in perfumery, cosmetics, toiletries, drugs and sanitation industries using plant according to claim 4.
Description:
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. .sctn. 119 to Indian Patent Application 202011026675, filed Jun. 24, 2020, which application is hereby incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 7, 2021, is named 217119-SeqList-6-7-2021.txt and is 45,056 bytes in size.
TECHNICAL FIELD
[0003] The present invention relates to a method for increasing viridiflorol content in tissues by overexpressing MPTPS4 through genetic transformation. This cultivar possesses better vegetative growth and is able to produce essential oil with good quality that contains high viridiflorol maximum of about 25% and possesses proportionate decrease in menthone, menthofuran and menthol of Mentha Piperita as compared to other control genotype(s).
BACKGROUND
[0004] Peppermint, Mentha piperita belongs to the family Lamiaceae. It is a natural hybrid between spearmint (Mentha spicata) and water mint (Mentha aquatica). The essential oil of Peppermint extracted via hydro-distillation method from the leaves and used in cosmetics, medicines etc. The essential oil is a mixture of terpenoids in varied proportions, and significantly finds application within the flavour or pharmaceuticals and oral medicative preparations. Comparatively small amounts of sesquiterpenes found in peppermint oil, thus there is an enormous gap within the area of sesquiterpene biosynthesis in genus Mentha that is very valuable and plays role in aroma value of oil. The main interest of terpene research is due to their diverse applications and utility with high market values in flavors and fragrances, medicines, industrial chemicals, and agricultural chemicals. Although, there is an immense demand for specific terpenes but the natural producers like plants and microbes synthesize terpenes/terpenoids/phenylpropenes/benzenoids in minute quantities. Chemical synthesis of these molecules is a costly and inefficient process. In addition, chemical synthesis leads to the formation of enantiomeric mixtures, adding problem and difficulties for separation and purification of desired molecule. Hence, with the ongoing efforts, there is also a need to explore new ways to enhance the production of these valuable classes of molecules. Viridiflorol is found to possess a green floral smelling aroma and possess anticarcinogenic and antitumorigenic properties but occurs in plants in very low amount. Supply of this molecule is limiting as these plants only grow in alpine region/endangered/difficult to multiply. Keeping in minds the importance of viridiflorol, the need for developing better plant type containing high viridiflorol content in essential oils of peppermint. In this invention an overexpression construct for the MPTPS4 under the tissue specific promoter mainly the trichome specific promoter (Limonene synthase) was designed as the essential oil is predominantly synthesized and sequestered in the glandular trichomes in Mentha Piperita and a maximum of about 25% viridiflorol could be obtained in the overexpressed transgenic plants & in heterologous system.
Objectives of the Invention
[0005] The main objective of the invention is to provide a method for obtaining tissues with high viridiflorol content to improve commercial value in terms of aroma as well as enhanced therapeutic properties.
[0006] Another objective of the present invention is isolate a sesquiterpene synthase (MPTPS4) gene from Mentha piperita and characterize functionally.
[0007] Yet another objective of the present invention is cloning and gene expression of MPTPS4, protein isolation and testing for conversion of Farnesyl di-Phosphate (FPP) to viridiflorol.
[0008] Yet another objective of the present invention is NMR analysis for confirmation of product of MPTPS4.
[0009] Still another objective of the present invention is validation of overexpression of transgenic plants by real time analysis and Gas chromatography-mass spectrometry (GC-MS).
SUMMARY OF THE INVENTION
[0010] Accordingly, the present invention provides a method for increasing viridiflorol content in tissues for improved commercial value in terms of aroma as well as enhanced therapeutic properties, as new class of compound will also add the aroma to the leaf of the plant and oil as compared to the non-transformed plants.
[0011] In an embodiment, a method for increasing viridiflorol content in tissues is provided. The method comprising:
[0012] a.) isolating sesquiterpene synthase gene (MPTPS4) from Mentha piperita having SEQ ID NO: 4;
[0013] b.) cloning of gene MPTPS4 obtained at step (a) into pHANNIAL vector to obtain a cassette with SEQ ID NO: 8;
[0014] c.) re-cloning the cassette obtained at step (c) into pART27 binary vector system with SEQ ID NO: 9; and
[0015] d.) transforming the vector obtained at step (c) into Agrobacterium tumerfaciens for overexpression of MPTPS4.
[0016] In another embodiment, a method for increasing viridiflorol content in tissues is provided, wherein the terminator sequence is an octapine synthase (OCS) terminator sequence is set forth in SEQ ID NO: 10.
[0017] In another embodiment, a method for increasing viridiflorol content in tissues is provided, wherein the promoter sequence is a tissue specific promoter (Limonene synthase, 797 bp) sequence is set forth in SEQ ID NO: 7.
[0018] In another embodiment, a product obtained by the method of the invention is provided.
[0019] The product obtained has increased viridiflorol content and is useful for perfumery, cosmetics, toiletries, drugs and sanitation products.
[0020] In another embodiment, a transgenic plant obtained by the method of the invention is provided. The plant obtained has increased viridiflorol content and is useful for products related to perfumery, cosmetics, toiletries, drugs and sanitation products.
[0021] The present inventors found that overexpression of (MPTPS4) produces high value viridiflorol synthase in Mentha under a GTS specific promoter. According to (Albert-Puleo, 1980, J. Ethnopharmacol. 2,337-344), Melaleuca quinquenervia (MQV) essential oil is rich in viridiflorol and could be applied to the whole body as it possesses male and female hormonal balancing properties which were mainly due to viridiflorol. The main attraction of overexpressing viridiflorol like molecule in Mentha Piperita is to enhance the medicinal properties as well as the aroma value of the plant. Viridiflorol possesses anticarcinogenic and antitumorigenic properties but occurs in plants in very low amount. The viridiflorol content is found to be higher in plants at higher altitudes. In this investigation an overexpression construct for the MPTPS4 under the tissue specific promoter mainly the trichome specific promoter (Limonene synthase) was designed as the essential oil is predominantly synthesized and sequestered in the peltate glandular trichomes in Mentha piperita. Viridiflorol is reported in essential oils of Mentha piperita up to (0.1-0.6%). In present invention, a maximum of about 25% viridiflorol could be obtained in the overexpressed the cells and tissues of the transgenic plants. This may be due to the trichome specific promoter which directs the expression of viridiflorol synthase gene in the glandular trichome, the active biosynthesis site and store house of terpenes.
[0022] Introduction of 4S-limonene synthase from Mentha spicata into Mentha arvensis and Mentha piperita quantitative and qualitative alterations were observed in monoterpenes pool in both species. Improvement of aroma and flavour in agricultural products by metabolic engineering is not sufficiently utilized. Overexpression of MPTPS4 in Mentha piperita increased the viridiflorol content up to 25% compared to 0.1-0.6% in cells and tissues of vector control plant. As observed in 5.4, Km of MPTPS4 is very low and hence utilizes the FPP pool in GTs efficiently. The utilization of FPP might have pulled the precursors IPP and GPP into the pool of FPP resulting into decreased supply of precursor to the monoterpene pathway. This present invention is not limited to the proportionate decrease in menthone, menthofuran and menthol but detected with the increase in viridiflorol. Further, similar pattern of increase in the content of the sesquiterpene alloaromadendrene like viridiflorol observed in all overexpressed lines. This may be due to the fact that increase in viridiflorol concentration might have been an advantage for some enzyme not necessarily biosynthesizing aromdendrene molecules but responsible for other function, to accept viridiflorol as a substrate (high Km) to convert a minor amount to Aromadendrine like molecules. Hence, though higher than control, the concentration of alloaromadendrine is very less in the transgenic plants for viridiflorol synthase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is schematic representation of preparation of overexpression construct.
BRIEF DESCRIPTION OF THE TABLES
[0024] Table 1 is viridiflorol analysis in essential oil of transgenic Mentha plants over expressing MPTPS4. Values are represented as mean.+-.SD (n=3 biological replicates), VC: Vector.
[0025] Table 2 is a comparative analysis of other metabolites in MPTPS4 overexpressing lines of Mentha in GC-MS (values are shown in percentage).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be illustrated in the following examples, which are in no way intended to limit the scope of the present invention.
Example 1
[0027] Expression of MPTPS4 Gene
[0028] 1. Selection of Genes to be Used
[0029] Comparative Transcriptome Analysis of Mentha piperita and M. spicata
[0030] To investigate the terpene synthesis pathway of Mentha Piperita recently constructed the Mentha piperita transcriptome through RNA sequencing of the GT's rich tissue the transcriptomes are deposited at NCBI under the accession no. SRP056511) (Akhtar, 2017 Physiologia Plantarum, 160:128-141) and compared with transcriptome of the GT's rich tissue of Mentha spicata (the transcriptomes are deposited at NCBI under the accession No. SRP056511) (Jin, 2014, BMC Plant Biology, 14).
[0031] In Silico Analysis of Database Sequences for TPSs Genes
[0032] In order to identify the valuable TPS genes, Plant trichrome EST database of Mentha piperita was mined to retrieve all the TPSs related transcripts/genes. Further, a local BLAST of these ESTs was carried out against the transcriptome database of Mentha piperita. This led to the identification of some uncharacterized TPS related genes. Based on nucleotide and amino acid sequences, partial ESTs were shortlisted and used to obtain full-length genes for further characterization.
[0033] Transcriptome Sequencing and Assembly
[0034] The sequencing of paired end cDNA library of transcriptome from trichomes of Mentha piperita and M. spicata generated a total of 412,458,378 (412.46 million) raw reads for Mentha piperita and 230,383,922 (230.38 million) raw reads for M. spicata. Out of which, number of paired end reads were 143,546,248 (143.5 million) for Mentha piperita and 81,145,014 (81.1 million) for M. spicata. Since, paired end sequencing generates high quality alienable sequence data and is more likely to align to a reference; these reads were used for further processing.
[0035] Assembly of Transcripts Showing Differential Expression
[0036] Transcripts from the libraries of both Mentha piperita and Mentha spicata were clustered using CD-HIT EST tool applying the criterion of sequence identity threshold as 0.8 for obtaining the set of non-redundant transcripts. A total of 52321 transcripts were obtained both from Mentha piperita and M. spicata out of which, 16138 transcripts were exclusive to Mentha piperita while, 896 transcripts were exclusive to Mentha spicata and 35287 transcripts were common in both Mentha piperita and Mentha spicata (456 up-regulated; 438 down-regulated and 34393 neutral with respect to Mentha piperita).
[0037] Functional Annotation and GO Classification
[0038] Annotations of all the unique transcripts (>300 bp) was performed using a BLASTx homology search against TAIR Database. BLAST hits with e-value scores .ltoreq.0.001 were considered as annotated homologous proteins. Transcripts were subjected to Gene Ontology (GO) classification in order to categorize them under the three categories of biological process (BP), molecular function (MF) and cellular component (CC). Out of the total 52321 transcripts in both plants, 36793 transcripts got annotated with different GO terms in aforementioned three categories of BP, MF and CC. The order of the GO term assignment was CC (68.61%)>MF (21.43%)>BP (9.94%).
Example 2
[0039] Identification of Transcripts Related to Terpene Synthases
[0040] To explore the array of terpenoids, it was important to identify the transcripts related to the terpene synthases. 89 and 82 TPS transcripts were identified in Mentha piperita and Mentha spicata, respectively when annotated against the TAIR database of Arabidopsis shows number of transcripts for each gene identified in the annotation.
TABLE-US-00001 TABLE 1 SAMPLE MARCH APRIL MAY JUNE JULY MPOT_1 8.66 +/- 0.0049 7.86 +/- 0.0015 8.71 +/- 0.0058 8.75 +/- 0.0025 7.65 +/- 0.0164 MPOT_2 25.98 +/- 0.0043 26.6 +/- 0.0045 25.2 +/- 0.0248 21.7 +/- 0.0341 24.43 +/- 0.0285 MPOT_3 20.40 +/- 0.0029 22.5 +/- 0.0038 19.2 +/- 0.0494 22.7 +/- 0.0107 15.14 +/- 0.0049 MPOT_4 14.53 +/- 0.0013 14.8 +/- 0.0065 14.2 +/- 0.0235 11.3 +/- 0.0198 12.43 +/- 0.0071 VC 0.40 +/- 2.30 0.56 +/- 0.0004 0.53 +/- 0.0001 0.54 +/- 6.67 0.42 +/- 0.0004
TABLE-US-00002 TABLE 2 Compound name MPOT_1 MPOT_2 MPOT_3 MPOT_4 Vector .alpha.-Pinene 0.59 0.54 0.58 0.60 0.69 .beta.-Pinene 1.44 1.23 1.32 1.38 1.61 D-Limonene 5.70 4.88 5.23 5.48 6.47 Linolool 0.21 0.18 0.19 0.20 0.23 Menthone 23.7 20.2685 21.95 22.924 27.394 Menthofuran 17.75 15.03 16.14 16.80 20.12 Menthol 18.95 16.14 17.36 18.16 21.71 .alpha.-Terpineol 0.66 0.59 0.62 0.65 0.78 Pulegone 3.38 2.89 3.17 3.29 3.97 Menthyl acetate 3.426 2.911 3.154 3.2865 3.93 .beta.-Bourbonene 0.10 0.09 0.09 0.11 0.12 Caryophyllene 3.04 2.59 2.79 2.93 3.47 .beta.-Cubebene 0.13 0.11 0.12 0.12 0.15 Alloaromadendrene 0.28 0.54 0.39 0.28 0.02 Humulene 0.19 0.16 0.17 0.18 0.21 cis-.beta.-Farnesene 0.38 0.34 0.35 0.35 0.38 Germacrene D 0.73 0.63 0.69 0.71 0.86 .gamma.-Muurolene 0.12 0.10 0.10 0.11 0.13 Viridiflorol 7.77 26.03 21.94 14.38 0.585
[0041] Pathway Analysis (Differentially Expressed)
[0042] MVA and MEP Pathway
[0043] Differential expression profile of transcripts annotated as MVEP and MVA pathway genes showed that the transcripts of genes involved in MVEP pathway namely CMK, DXR, DXS, MCT, MCS, HDR and GGPP are upregulated in Mentha spicata and MVA pathway transcripts namely AACT, HMGS, WVK, PMK, PMD and FPP are downregulated compared to the reference gene ACT (Actin).
[0044] Terpene Synthases
[0045] In the differential gene expression analysis, a total of 94 TPS transcripts were found, out of which, 12 were up-regulated, 9 were down-regulated and 44 were neutral in Mentha spicata when compared to Mentha piperita, while 11 TPS transcripts were exclusively expressed in Mentha piperita and 18 were in Mentha spicata.
[0046] Analysis of Available Mentha piperita Trichome EST Database for Terpene Synthase
[0047] ESTs analysis of the Plant trichOME database revealed the presence of about eight different TPS ESTs.
Example 3
[0048] Identification, Isolation and Sequence Characterization of the TPSs in Mentha piperita
[0049] A local BLAST of 45 ESTs from Plant trichOME database was performed against transcriptome database of Mentha piperita. Out of these 11 ESTs which were found less annotated to the database were used to get full-length genes. RACE primers were designed from the available TPS partial sequences to obtain full-length genes. A total of six genes namely MPTPS1 with SEQ ID NO: 1; MPTPS2 with SEQ ID NO: 2; MPTPS3 with SEQ ID NO: 3; MPTPS4 with SEQ ID NO: 4; MPTPS6 with SEQ ID NO: 5 and MPTPS7 with SEQ ID NO: 6 were cloned from Mentha piperita. All the genes were amplified from the cDNA of trichome rich tissue of Mentha piperita (CIM-Madhuras), cloned, sequenced and characterized. The sequences obtained were subjected to BLASTx and BLASTn analysis. The amino acid sequence of Mentha piperita was compared to ones existing in the NCBI database for the identification of signature sequences/motifs and or domains.
[0050] Out of these six terpene synthases, four (MPTPS1, MPTPS2, MPTPS3, and MPTPS4) are sesquiterpenes and two (MPTPS6 and MPTPS7) are monoterpenes. In sesquiterpenes, MPTPS3 and MPTPS4 showed less identity to others from the database after BLASTx analysis, therefore, MPTPS3 and MPTPS4 were considered for further analysis and characterization.
[0051] MPTPS3 and MPTPS4 Gene Expression in Different Tissues.
[0052] Comparison of the deduced amino acid sequences of MPTPS3 and MPTPS4 showed 81% similarity. Tissue-specific expression was analyzed for MPTPS3 and MPTPS4 in different tissues in Mentha piperita for comparison. MPTPS4 showed significantly higher spacial expression than MPTPS3 in the order of trichome>leaf>stem>root. Hence, only MPTPS4 was taken into consideration for the further downstream functional characterization.
[0053] MPTPS4
[0054] EST of MPTPS4 was extracted from the plant trichOME EST database of Mentha piperita annotated as Terpene synthase (Accession no AW255698). The partial sequence thus obtained was 628 bp and which was subjected to 3' and 5'RACE in order to get the full-length MPTPS4 gene. Full-length cDNA sequence of MPTPS4 gene was found to be 1686 bp, encoding a polypeptide of 561 amino acid residues. The molecular weight of the deduced protein was predicted to be 65.09 kDa and computed isoelectric point (pI) was 4.91. The nucleotide sequence has been submitted to the NCBI database with the accession number (MH790402). Nucleotide sequences of MPTPS4 from Mentha piperita showed 82% similarity with TPS4 of Origanum vulgare, while amino acid sequences showed 77% sequence similarity with TPS4 of Origanum vulgare which is reported to be a Bicyclogermacrene synthase.
Example 4
[0055] Cloning of MPTPS4 Gene in pET28a (+) and Transformation for Bacterial Expression
[0056] The MPTPS4 gene (.about.1.6 kb) was sub cloned from pGEM-T Easy cloning vector onto the pET28a (+) expression vector. The positive recombinant plasmid was then transformed 30 into E. coli BL21 (DE3) bacterial expression host cell and both the induced and uninduced host proteins were isolated to analyze on the SDS PAGE. 64 kDa MPTPS4 protein was obtained after purification and desaltation from the induced host cell was confirmed by running SDS PAGE and comparing with the protein molecular weight marker.
[0057] Enzyme Assay
[0058] GC-MS Analysis
[0059] The recombinant MPTPS4 protein was produced in E. coli BL21 (DE3), enzyme assay reactions were set up with crude extracts in which extract was incubated with FPP and the product was analyzed in GC, a unique peak of viridiflorol was observed when compared to the vector control. Similarly, purified MPTPS4 protein was incubated with enzyme substrates FPP, GPP and GGPP and the reaction products were analyzed using GC-MS. The reactions in which GPP and GGPP were used as substrates did not show any product formation. On the other hand, when FPP was used as the substrate of MPTPS4 protein, a product peak was generated. The product was identified to be "Viridiflorol" based on the mass spectra similarity as per the NIST library.
[0060] Enzyme Kinetics
[0061] The purified recombinant MPTPS4 enzyme was characterized for their catalytic activities with FPP. Rate of reaction for different substrate (FPP) concentration (0-350 .mu.M) (was also analyzed). The amount of substrate converted in each reaction was calculated, and the amount of enzyme was standardized to micromole per minute per microgram. The reaction rate per microgram enzyme per second was established, and the reaction rate (1/second) was calculated.
[0062] Product Confirmation of MPTPS4 Through NMR Studies
[0063] Structural Analysis of MPTPS4 Product
[0064] The expression construct pET28a-MPTPS4 was introduced into E. coli BL21-CodonPlus (DE3) cells together with plasmid Add gene Plasmid 35150: pBbA5c-MevT-MBIS, for the utilization of FPP as the substrate, were grown in LB medium and the product extracted was 25.2 mg. This product was subjected to .sup.1H-nuclear magnetic resonance spectrometry, .sup.13C NMR and DEPT 135 spectrum. The .sup.1H and .sup.13C chemical shift of viridiflorol is shown in GC-MS and TLC analysis.
Example 5
[0065] Preparation of Over Expression Construct of MPTPS4
[0066] The MPTPS4 (1686 bp) gene with SEQ ID NO: 4 was cloned in pHANNIBAL vector system by replacing the PDK intron to yield phannibal-MPTPS4, under tissue specific promoter (Limonene synthase, 797 bp) with SEQ ID NO: 7. The whole cassette (.about.3.5 kb) SEQ ID NO: 8 was cloned into pART27 binary vector system with SEQ ID NO: 9 and confirmed by restriction analysis. The binary vector with and without the genes were then transformed into GV3103 strain of Agrobacterium tumefaciens separately, positive clones selected on kanamycin antibiotic plates and confirmed with the help of colony PCR.
[0067] Generation of Transgenic Mentha Lines of MPTPS4
[0068] In vitro maintained Mentha plants in the laboratory were used for Agrobacterium mediated internode transformation for generating transgenic plants. All individual transformation experiments were accompanied with vector control. PART-MPTPS4 transformed internode was placed on selective MSB_M media. After 3-4 weeks, direct regeneration was induced as a result of pART-MPTPS4 gene transformation on kanamycin (50 mg L.sup.1) selection medium.
[0069] Analysis of Transgenic Mentha Plants
[0070] PCR analysis was carried out to confirm the transfer of transgene cassette into the transgenic lines. Genomic DNA was extracted from each putative transgenic line and PCR analyzed using primers of npt II gene. Four lines of pART-MPTS4 exhibited an amplification of 750 bp, which was absent in the no template control lane, hence were confirmed to be transgenic lines.
[0071] Overexpression Studies Through Quantitative Expression
[0072] Four independent kanamycin-resistant Mentha piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) and pART only (Vector Control, VC) transformants were grown in green house. The expression levels of the transgenic lines were determined by quantitative RT-PCR. Overexpression in four independent transgenic lines was in order of MPOT_2 (18.9-37.7 fold)>MPOT_3 (13.4-26.4-fold)>MPOT_1 (9.4-25 fold)>MPOT_4 (5.4-16.0 fold) compared to the vector control. The range denotes in planta fold expression as estimated from the isolated mRNA during March to July.
[0073] Overexpression Analysis Through GC-MS
[0074] In order to study the effect of increased MPTPS4 expression on the content of plant essential oils, four transgenic (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) plant lines produced through Agrobacterium transformation were grown in a greenhouse and essential oil profiles for all these lines were generated using GC-MS. All the four MPTPS4 over-expressed transgenic lines exhibited significant increase in viridiflorol content compared to the vector control. The range denotes in planta fold expression as estimated from the isolated mRNA during March to July.
[0075] Correlation of Metabolite Synthesis with Gene Expression
[0076] To see the effect of MPTPS4 overexpression on the other metabolites, comparative analysis was performed through GC-MS and values (%). All the four MPTPS4 overexpressing transgenic lines exhibited increase in the viridiflorol content while, other metabolites like menthone, menthofuran and menthol decreased as flux diverted towards increased content of viridiflorol as well as one sesquiterpene, alloaromadendrene also increased compared to control plants.
[0077] Subcellular Localisation of the MPTPS4
[0078] N-terminal in-frame GFP fusions were made with the MPTPS4 full-length coding regions in the p326-SGFP vector between the XbaI and BamH I restriction enzyme sites to create p326-MPTPS4/SGFP. GFP fluorescence for full-length p326-MPTPS4/SGFP coding regions was observed as a diffused signal exclusively in the cytosol. Expression of a control GFP construct was also localized in the cytoplasm. These results confirm the predicted cytosolic localization of the enzyme.
[0079] Procurement Details of all the Biological Materials Used in Invention
[0080] Mentha plants: The germplasm accessions of Mentha piperita cv. `Cim-Madhuras` was obtained from National gene Bank for medicinal and Aromatic Plants (NGBMAP) maintained at Central Institute of medicinal and Aromatic Plants (sponsored by Department of Biotechnology, Regime of India) in Lucknow (26.5.degree. N, 81.010 E), India.
TABLE-US-00003 Bacterial Host Strains Strain Description Reference Escherichia supE44 .DELTA. lacU169 (.phi.80 lacZ .DELTA. M15) hsd Sambrook et al., 1989 coli R17 rec Al endA1gyrA96 thi-1 rel A1 (DH5.alpha.) Escherichia F.sup.-ompT hsdSB(r B.sup.- m B.sup.-) gal dcm lacY1 Novagen coli (DE3) www.novagen.com BL21 Star .TM. (DE3) Agrobacterium Resistance for gentamycin and rifampicin Intact Genomics tumefaciens antibiotics www.intactgenomics.com (GV3103)
TABLE-US-00004 Vectors Plasmid vectors Description Source pGEM .RTM.-T 3.0 kb in size, multiple cloning Promega sites having 15 unique www.promega.com restriction sites, LacZ fragment, pUC/M13 forward and reverse priming sites, T7 and SP6 promoter/primer binding site, fl origin, T7 RNA polymerase transcription initiation site and has ampicillin resistance ORFs. pET 28a(+) 5.369 kb, N-terminal His.cndot.Tag .RTM. Novagen configuration with an optional www.novagen.com C-terminal His.cndot.Tag sequence, f1 origin of replication, T7 promoter, T7 terminator, lacI coding sequence, kan coding sequence, f1 origin replication. pHANNIBAL 5.824 kb in size with bacterial CSIRO ampicillin resistance is www.pi.csiro.au designed for directional insertion of PCR products on either side of the PDK intron. pART 27 Size of 11.667 kb, RK2 CSIRO minimal replicon for www.pi.csiro.au maintenance in Agrobacterium, the ColE1 origin of replication, Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker, nos promoter, nos terminator, LacZ fragment p326-sGFP 4.488 kb in size, CaMV35S Provided by Inhwan Hwang promoter, nos terminator, (POSTEC, Korea) ampicillin resistance, 900 bp sGFP genes cloned between the promoter and terminator. pBbA5c 1.3264 kb in size, lacUV5 Addgene promoter, rrnB terminator, https://www.addgene.org chloroamphinicol resistance, low copy.
[0081] pART-MPTPS4: --The MPTPS4_sense gene (500 bp) and MPTPS4_antisense gene (500 bp) were subcloned from pGEM-T Easy cloning vector onto the pHANNIBAL cloning vector under tissue specific promoter by replacing cauliflower mosaic virus (CaMV) 35S promoter with octopine synthas (ocs) terminator with SEQ TD NO: 10, sequentially on either side of the intron between EcoRI/KpnI and XbaI/HindIII restriction enzyme sites respectively to get the two arms of the hairpin which was confirmed by restriction analysis using the XhoI and XbaI, the first and the last restriction enzyme sites of multiple cloning sites, MCS1 and MCS2 of pHANNIBAL respectively. The hairpin cassette was then cloned into a pART 27 binary vectors in a single step using the Not I restriction sites as there are two NotI restriction enzyme sites in pHANNIBAL and a single site in pART27 binary vector.
[0082] cDNA of trichome rich tissue of Mentha piperita (CIM-Madhuras):--cDNA of trichome rich tissue of Mentha piperita were isolated in Dr. Ajit K. Shasany's Lab, Biotechnology Division, CSIR-CIMAP, Lucknow, India.
[0083] Kanamycin-resistant Mentha piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4):--To prepare the overexpression construct of the MPTPS4, specific primers with SEQ ID NO: 11 and SEQ ID NO: 12 were designed introducing BamHI with and EcoRI with restriction enzyme sites by replacing the PDK intron into pHANNIBAL vector system to yield pHANNIBAL-MPTPS4 under tissue specific promoter by replacing cauliflower mosaic virus (CMV) 35s promoter with octapine synthase (OCS) terminator. The cloned fragments were confirmed by restriction analysis and sequencing. The whole cassette with promoter, gene and terminator was then cloned into pART27 binary vector system. The cloned fragments were confirmed by restriction analysis. Binary vector with and without the transgene (MPTPS4) was then transformed into GV3103 strain of Agrobacterium separately.
[0084] Generation of transgenic Mentha lines of MPTPS4--In vitro maintained Mentha plants in the laboratory were used for Agrobacterium mediated internode transformation for generating transgenic plant. All individual transformation experiments were accompanied with vector control. PART-MPTPS4 transformed internodes were placed on selective MSB_M media. After 3-4 weeks, direct regeneration was induced as a result of pART-MPTPS4 gene transformation on kanamycin (50 mg L.sup.1) selection medium. Four independent kanamycin-resistant Mentha Piperita transformants of pART-MPTPS4 (MPOT_1, MPOT_2, MPOT_3 and MPOT_4) and pART only (Vector Control, VC) transformants were obtained and grown in green house. Experiments were performed in Dr. Ajit K. Shasany's Lab, Biotechnology Division, CSIR-CIMAP, Lucknow, India.
Advantages of the Invention
[0085] The present invention provides for:
[0086] 1.) A method for obtaining transgenic plants with high viridiflorol containing variety for enhancing the aroma, therapeutic properties.
[0087] 2.) A method for enhancing the medicinal properties of Mentha piperita by overexpressing MPTPS4 through genetic transformation.
[0088] 3.) The method provides better vegetative growth.
[0089] 4.) The method produces plants with essential oil with good quality containing high viridiflorol maximum of about 25%.
[0090] 5.) The plants obtained with increased viridiflorol content produced by the method of the invention are used in perfumery, cosmetics, toiletries, drugs and other sanitation products.
[0091] 6.) The produced viridiflorol adds sweetness to the aroma of plant essential oil.
[0092] 7.) The obtained viridiflorol possess anticarcinogenic and antitumorigenic properties.
REFERENCES
[0092]
[0093] 1. Albert-Puleo M (1980) Fennel and anise as estrogenic agents. J Ethnopharmacol 2:337-344.
[0094] 2. Akhtar M Q, Qamar N, Yadav P, et al (2017) Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species. Physiologia Plantarum 160:128-141. doi: 10.1111/ppl.12550.
[0095] 3. Jin J, Panicker D, Wang Q, et al (2014) Next generation sequencing unravels the biosynthetic ability of Spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics. BMC Plant Biology 14. doi: 10.1186/s12870-014-0292-5.
Sequence CWU
1
1
1211677DNAMentha piperitagene(1)..(1677)The partial sequence thus obtained
was 447 bp and was incomplete from the 3' region. 3' RACE was
carried out to convert it into a full-length gene. Full-length cDNA
sequence of MPTPS1 gene was found to be 1680 bp, encoding a a
polypepeptide of 560 amino acids. 1atggctgaaa tctgtgcgtc ggctgctcca
atctcaacaa agaatacaag tgtagaggaa 60atcctcgatc ggtaacatat catcccagcg
tttggagaga tcattttctt gcatatacta 120acgacgtcac ggaaatcagt gctgctgaga
aggaacaact cggaaagcaa aaggaaaagg 180ttaagaattt gctactcaaa ctccaaatga
ttcaacgctc aagatcgacc tcatcgatgc 240aatccaacgt ctagggttgg gctatcattt
cgaggaggaa atcgacggat ccttgcgaaa 300aattcgcgac agttatgaaa tgttaagtag
caaaggcgag ggcgatgtcc gtgttcttgc 360tcttcgcttt cgtctgctta gacaacaagg
ttatcgcgtc ccatgcgaag tgttcaacaa 420attggtagac gacgaaggga attttaagga
gtcgttgatt aacgacgttg aagggatgct 480aagcttgtac gaagcttcga attatggaat
aaatggagag gaaattatgg acaaagcctt 540agaattttct tcttctcatc ttgaggattc
aatccaaaaa aaacccactt gtctttcgag 600acgagtgaaa gaagctttgg atatgccgat
tagcaagact ttgatgagat tgggagcgag 660aaaattcata tctatttatg aagaagacga
gtcgcataat gaattattat tgaattttgc 720aaaactggac ttcaatatag tacaaaagat
gcatcaaaga gagttgcacc atattacaag 780gtggtgggaa gatttagact ttaaaagtaa
actacctttt gcaagagata gagtggtgga 840gtgctacttc tggattttgg gagtttattt
tgaaccaaaa tacgaaactg caagaagatt 900actaactaaa gtcatatcca tggcttccat
ccttgatgat atatatgatg tttatgggag 960tttagatgaa ctccgacatt tcaccgatgc
tattcaaaga tgggatattg tcggtgctga 1020agagttgcca ccatacatga gaatatgtta
tgaagcttta ctaggtgtat atgccgaaat 1080ggaagatgaa atggtaaaac aaggccagtc
atatcgcata gtctatgcaa aacaggagat 1140gataaaattg gtgagggcgt atatggaaga
ggcggagtgg tgttatagca agtatattcc 1200aaggatggat gagtatatga aactggcact
cgtatccggc gcttacatga tgctatcgac 1260aacttcttta gtcgggatga tggaagaacc
catctcagta cacgattttg attggatcac 1320cggcgaacca cccatcttac gagctgcatc
cgttatttgt aggttgatgg acgacatggt 1380aggccatggg attgagcaaa aaattacgag
cgtggatatt tacatgaggg aaaacgggtg 1440ctcgaagacg gaagcttttg gagagttttg
gaaacgagtg aagaaagcat ggaaggatat 1500gaatgaggag tgcctggagc caaggccagc
atccatgccc atacttactc gagttctcaa 1560tcttgctcgc gtcatcaatt tattgtacgt
tggtgaagat gcctatggta gttcaagtac 1620taagaccaaa gatttcattc aatccgtgct
cgtagatccc ctgcactcaa ctatttg 167721635DNAMentha
piperitagene(1)..(1635)The partial sequence obtained was 420 bp and
was incomplete from the 3' region. Hence, 3' RACE was carried out to
convert it into a full length gene. Full-length cDNA sequence of
MPTPS3 gene was found to be 1686 bp, encoding a polypetide of 560
amino acids.gene(1)..(1635)The partial sequence thus obtained was of 420
bp which was further subjected to 3' and 5'RACE in order to get
the full-length MPTPS2 gene. Full-length cDNA sequence of MPTPS2
gene was found to be 1635 bp, encoding a polypeptide of 545 amino
acids. 2atggatattc ctgcaccagt ttcggcttac aacgacgacg ttcgacgctc tgtaacttac
60catcccaaca tttggggaga cttttttctt gcctatactt cagacttcac ggaaacctcc
120attgctgaaa aggaagaaca cgaaaggcta aaggaggaga ttaggaaatt gctcctgcaa
180actgaagatg aatcaacgct gaaattagaa ctcatcgatt cagttcaacg ccttggagta
240ggctaccatt tccgaaaaga aatcgataca gcgttgcgtt acgttcatga caccaagaac
300aaggacgatc ttcgtgtagt agctcttcgc tttcgcctcc ttagacaaca tggtttccat
360gttccttgcg atgtgtttag tgaattcata gacccccaag gaaatttcaa ggagtcgata
420atcaacgatg ttgaagggat attgagctta tacgaggcat caaattatgg agtgcatgga
480gaagaaattc ttgacaaagc attaggtttt tgttcatctc gtctcgaatc tttagtcacc
540gacatgaatg atacttgtct ttcaagacaa gttaaggaag ctttgaagat cccaattagc
600aagactctaa cgaggttggg agcaagaaag ttcatttcta cgtatcgaga agtcgactca
660cacaacgaaa aattactcaa ctttgccata ttggacttca acctagtaca aaggctacat
720cagaatgagc tcagccatct tacaaggtgg tggaaggaat tagactttgc aaataagcta
780tcttttgcta gagatagact agtggaatac tatttttgga ttgtgggagt ttattttgaa
840ccgcggttcg gtattgcacg aaaattacta accaaagtca tttatatggc ttccgtcctt
900gatgacattt acgacgtgta tggaactctg gacgaactaa tgcttttcac gagcattgtt
960cgaaggtggg acattagtgc tattgatcaa ttgcctgcat acatgagaat atacttgaaa
1020gccctcttcg atgtgtatgt tgaaatggaa gaagaaatgg gaaagatagg caaatcatac
1080gcaattgaat atgcaaaaga agagatgaaa agattggcgg agatgtacct tgaagaggca
1140aaatggtcct ttagcaagta caagcccaca atgcgagagt acatgaaggt ggctctttta
1200tcatccggtt acatgatgat gacagtaaat tcattagctg ttatcgaaga tgaaattacc
1260aatgaagagt ttgattgggt tttgagcgaa ccgccaattc taaagtcatc gttgatgatt
1320acaagattaa tggacgacct tgcaggatat gggtttgaag agaaaaactc agcagtgcat
1380tactacatga atgaaaaggg cgtgtcggag ggggaagcca ttgcggagtt ccggaaacaa
1440gtggagaagt catggaagac tctaaacaag gaatgcttag agccaagagc agcctccatg
1500cccatcctca agtgtgttgt gaattttact cgtgtcatag ttgtgttata cacagatgaa
1560gatgcatacg gaaattccaa aactaaaacc aaagatatga tcaaatccat actcgttgat
1620cccctaacag tttag
163531686DNAMentha piperitagene(1)..(1686)The partial sequence obtained
was 420 bp and was incomplete from the 3' region. Hence, 3' RACE was
carried out to convert it into a full length gene. Full-length cDNA
sequence of MPTPS3 gene was found to be 1686 bp, encoding a
polypeptide of 560 amino acids. 3atggaaattt attccccagt tgttcctgca
actgcaatca aagatgtgaa gaagttgaat 60gaaattcgaa aatccgccaa attccatcca
actatttggg gagactattt tctagcttat 120aattctgata atacgctaac ctcaaatggc
gaagaaaaag aagttgcaaa gcagaaagaa 180atggtgcgga aactgctagc tgaagcccca
gaaaattcca cctacaaaat ggaactcatc 240gatacaatcc agcgcctggg agtggaatat
cattttgaaa aagaaattga aaattcctta 300aaatatattc ataaaaatta tgtccaccaa
aatagtaaag acgacgatcc ccacaccgtg 360gctcttcgtt ttcgtttgct cagacaacaa
ggttacaacg tcccatgcga tgttttccgc 420aaattcattg atagtgaagg aaattttatg
gagtcatcga cagagaacgt tgaaggtttg 480ttggaattat acgaggcatc gcatctcgcg
acacgtggcg aggaaattct gaatagagca 540atggagtttt gttcttccca tctccaaaca
ttagtgaatc agcagttagt agaaaatgtt 600tctctttcta aacgtgttag cgaagctctg
aagatgccaa tttgcaggag tcttacaagg 660ttgggtgcca gaaagttcat ctctctatac
gaagaggatg attcgcacaa tgaaatactt 720ttgaattttg caaaattgga tttcaatatt
gtgcagaaga tgcaccagag agagctcagt 780gatgctacta ggtggtggaa aaaattggag
gtggcgaata aaatgcctta cgcgagagac 840agaattgtgg agtgcttctt ttggatagtg
ggggtctact ttgagccatg ctattctatt 900gcaagaagaa tattaatcaa aagcataagt
atggcttcca ttattgatga cacctacgaa 960tatgcaaccc tagatgaact gcaaattcta
actgatgcta ttcaacgttg ggatgttaac 1020gagacattgg aagattcgcc accgtacatc
caaatgtgct acagaagcct tatccaagct 1080tatgctgaaa tagaagatga agtactgaag
aagaactcag aagaatcgta ccgcgtccaa 1140tatgcaatac aagatatgaa aaaattggtg
atggcatatt atgaagaggc gaaatggttg 1200tacaataata gtattccaac aatggaggaa
tatatgaagg tgtcactagt ttcatgtggt 1260tacatgatgt tgtcaacaac ttctttagtt
ggtatgggga ctaatcaagt tagcaaatca 1320gattttgatt ggattgtaaa tgaacctcta
atggttcgag catactcagt aatttgtcga 1380ctaatggacg acttagtcgg agacgagtat
gaggagaagc cgtcgtcggt ccattgttac 1440atgaagcaat atggaatgtc aaaggaggaa
gctcgagctc aactcgaaga acaagtgaaa 1500ggagcatgga aggatatgaa tgaagaatgc
ctcgagccga gaccagcctc gatgcaaatc 1560cttatgcgcg ctttgaattt tggtcgagtc
ataaatcttc tgtatgcaga agacgattgc 1620tatgccaatc ccattaattc caaagaatgg
gtgaagatgg tgcttgttga gcctgttccc 1680atttga
168641686DNAMentha
piperitagene(1)..(1686)The partial sequence thus obtained was 628 bp
and which was subjected to 3' and 5'RACE in order to get the full-
length MPTPS4 gene. Full-length cDNA sequence of MPTPS4 gene was
found to be 1686 bp, encoding a polypeptide of 561 amino acids.
4atggaaattt attccccagt tgttcctgca atcaaagatg tgaagaggtt ggatgaaatc
60cgaaaatccg ccaaatacca tccaactatt tggggagact tttttctagc gtataattcc
120gataatacga aaatctctga tggtgaagaa gaagaagttg caaagcagaa agaaatggta
180cggaaactgc tagctgaagc cccagaaaat tccacctaca aaatggaact catcgataca
240atccagcgcc tgggagtgga atatcatttt gaaaaagaaa tcgaaatatc cttgaaatat
300attcatgaga attatgtgca ccaaaacagc aaagacgacg atcttcacac cgtggctctt
360cgttttcgtt tgcttagaca acaaggttac aacgtcccat gcgacgtttt cagcaaattc
420accgaccgtg aaggaaattt tgcggcggcg ctgagaaatg acgttgaaag tttgttggaa
480ttatatgagg cgtcgcatct cgcgacacgt ggcgaggaaa ttctggacag cgcaatggag
540ttgtgttctt cccatctcca agcattagta aatgatcagc agttggtgaa caatgtttct
600ctctctaaac gagtgattga agctctgaag atgccaattc gcaagagtct cacgagattg
660ggtgcgagaa agttcatctc tctataccaa gaggatgatt cgcataatga aatacttttg
720aattttgcga aattagattt caatatagtg cagaagatgc accagagaga gcttagtgat
780gctacgaggt ggtggaagaa gttggacgtg gcgaataaaa tgccttacgc aagagacaga
840aatgtggagt gcttcttttt ggatggtggg cgtctacttc gagcatgcta cgctactgca
900agaaaaatat tacttaaatg cataagtatg gcttcaatta ttgatgacac ctacgaatat
960gcaaccctac atgaactgca aattctcacc gacgctatcc aacgttggga tgttaatgag
1020gcattggagg attcgccacc atatatacaa atgtgctaca gaagccttat tcaaacttat
1080gttgaaatag aagatgaagt agtggagaaa tttggaggag aatcgtcata ccgtgtccaa
1140tatgcaatac aagatatgaa aaaatcggtg tgggcatata tggaagaggc gaaatggatg
1200tatgacgatt atattccaac cgtggaggag tatatgaagg tttcgatcgt atcttgtggt
1260tatatgacaa tgtcaaccac ttctttagtg ggtatgggga tcgatcaagt ttgcaaagaa
1320gatttcgatt ggatcgtaaa tgaacctctc ttggttcgag cttcatcggc aatttgtcga
1380ttaaccgacg atctagtcgg ggacgagttt gagcaaaaaa cgtcgttggt ggattgttac
1440atgaaacaac acggtatgtc gaaggacgaa gctcgagctc atcttcgaca acagttgaag
1500gatgcatgga aggatatgaa ccaagaatcc cttgagccta gaccggcctc gatgccaatc
1560ctaacgcgtg ttatcaatct cggccgagtc atagatcttc tctattcgga ccatgattgc
1620tatagtgatc ccaacaaatc gaaaggttgg gtaaagatgg tgctcgtcga ccccatcgca
1680atttga
168651816DNAMentha piperitaGene(1)..(1816)The partial sequence thus
obtained was 759 bp and which was subjected to 3' and 5'RACE in
order to get the full- length MPTPS6 gene. Full-length cDNA sequence
of MPTPS6 gene was found to be 1821 bp, encoding a polypeptide of
607 amino acids. 5atgtccacca ttataatggg catgacgctt cccaacaaac ctaccatttg
tgttgataac 60ttcgcaacta aatatccaaa tctgcgccga gcttttccgg tttcatgccg
ccgccgtcag 120tcttccgccg tcaaactcag cgctaacact gcttgtacag atgaactcca
atctacaaga 180cgatcgggaa attacgaacc taccctatgg gattttgatc gtattcagtc
actcaatagt 240gtttgcacgg agagggacgg aagaagggca gcggttttga taaaggaagt
gaagatgttg 300ttacaggaag aagtggatgg tgttcttcga cagctggagt tgattgatgc
cttgcagagg 360ctgggtatat cttgtcactt caatgaagaa atcaaacaaa tcttgaattc
tttttattac 420aacgagttca atgatgcaat agttgcagag gaaagggatt tgtacttcac
agctcttgca 480ttcagactac tcagacaaca cggttttaac gtctctcaag aagtctttga
ctatttcaag 540agtgaagagg gtattgatga tttcaagacc atccatgctg aagataccaa
agggctgtta 600caattgtacg aagcatcttt tctatcaaca caaggcgaag aaaccctaga
attagcaaga 660gaatatgccc taaaattttt gcagaaaata ctcgatcatg aaattattaa
tgacgaaaat 720ctctcatcat caattcttcg agatgccata aaaatcccca ttcactggag
ggttcaaatg 780ccaaacgcaa gatcctatat tgatgcctat gagaggaaac ctcgcatgca
tccaattgta 840ctagagctag ccaaactaga gataactatt gttcaagcac gggttcaaca
agaactcaaa 900gagacctcaa ggtggtggca tagcacgagc ctcgttcaac aacttcccct
tgtgagggat 960aggatcgtgg agtgctacta ttggacgacc ggagtccttg agcgtcgaga
acatggatat 1020gagagaataa tgctcaccaa aataaatgct cttgttacaa ctattgatga
tatttatgat 1080atttacggca catttgaaga gctccaacta ttcactaacg cgattaaaag
atgggatata 1140gaatcgatga atcaactacc tccttacatg caacaatgct atcttgcact
ccaaaatttt 1200gttaatgaga tggcttacaa taccctcaag caaaaaggct tcaactcaat
cccatatcta 1260cataaaacgt gggttgattt ggttgaggca tatatgagag aggcagaatg
gtaccgcaac 1320ggtcataaac ctagcctcga agaatatatg aataatgcat ggatatcaat
cggaggcgtc 1380ccgattttat cccatatctt tttctgtgta acagattcta tagatgaagt
gaccgttgag 1440agggtgcatg aataccatga tatagctcgt gcatcatgta cgattcttag
gcttgctgat 1500gatttgggaa catctttgga tgaggtgaag agaggagacg taccgaaatc
agttgaatgt 1560tacatgaatg atgaaaagaa tgcttctgaa caagaggcgc gggcgcatgt
acgatctatc 1620ataaagaata catggaaaac gatgaatgag gaaatgatga cgtcaactaa
ttctcaattt 1680tcaaaatatt ttgtggaagc tgctgctaat cttgggagaa tgtcactttg
tatctaccaa 1740gatgaatgtg atggcttcgg catgcaacat tcaagggtta acaaaatgct
aagaggcttg 1800ctcttcgacc cctgta
181661812DNAMentha piperitagene(1)..(1812)The partial sequence
thus obtained was 401 bp and was subjected to 3' and 5' RACE to get
the full-length MPTPS7 gene. Full-length cDNA sequence of MPTPS7
gene was found to be 1812 bp, encoding a polypeptide of 604 amino
acids. 6atgtctaccc ttagcatgca cgtatcgatc tttagcaagc aagttgaaaa taacaacttc
60ttgggcatga gagcttcaaa accaatggcg agacgcgtag cttccactcg cctccgccct
120atttgctccg ccaccgccac acaaccacaa gttgaagaaa cccgacgttc tggaaactac
180caggcttcca tttgggactt caattacatt caatctctcc atactccaga ctacaaggaa
240gagagacttt tgaataggaa agaagagctg atcgtggagg tgaagaagtt gttgaagggg
300aaaatggagg cagctaagca gttggagctg atcgatgact tgcagaattt gggattgtct
360tatttttttc aagacgagat taaaaatatc ttgaattgta tatataatga gcacaattat
420ttccaaaata atattagtaa agtaggggat ttgcatttca ctgctcttgg attcagactc
480ctcagacagc atggtttcaa cgtttcacaa ggagtatttg agtgtttcaa gaatgaggag
540ggtagtgatt tcgaggaaac cctaattggg gaagatacga aaggaatatt gcaactttac
600gaagcatctt tccttttgag agaaggtgaa gatacacttg agctagctag aaaattctca
660accaaatatc tgcggaaaaa agttgatgag ggaataataa atgatgataa taatctgtta
720tcatggattc gtcattcttt ggatctccct cttcactgga ggattcaaag actcgaggca
780agatggttct taaatgctta ctcaaggagg aaagacatga atccacttat tttcgagctc
840tcaatactcg acttcaatat cattcaagca acacacatac ttgagctcaa agaggtctct
900aggtggtgga atgaatcggg ccttgttgag aaactaccct ttgtgagaga taggttggtg
960gaaagctact tttgggccct tggccttttc gaggcccatg attatggata tcagagaaaa
1020attacaacct taattattgc tctaatcacg gctaccgatg atgtttacga tgtttatggt
1080acattagacg aactccgctt atttacagat gcgattcgaa gatgggatac aaaatcaata
1140gaccaacttc cgtattacat gcaactctgc tatttggcac tccacaactt agtttccgag
1200atggcttacg attgtctcaa acataaacag ttcaacagta ttccatattt tcagcgatcg
1260tgggtgagtt tgggggaagg atatttgaaa gaggcagagt ggttcgaaag tggatacaca
1320ccaaccctag atgaatacct caacaacgcc aagatttcaa taggctctcc cacaataatc
1380acccaagttt acttaacctt actaaactcg agcaccgacc aacaagtcct tgatagcttg
1440tacgagtacc acaacatact ctatctttcc ggtatagttt taaggcttgc tgacgatcta
1500ggtacatcac agtttgagct gaagagaggg gacgtaccaa aagcgttgca gtgctgcatg
1560aaggacatga aatgtaggga gaaagaggcg gaagaacacc tgcggtttct gatccgagag
1620gcgtggaagg agattaacac aacgatgggg gcggaagctg cagattgtcc gtacaaagat
1680gatttgtttg aggctgcggc taaccttgga agagctgctc agtttatata tttggaggga
1740gatggtcatg gcgttcagca ttccggaata catcaccaga tggccagctc aatgttccac
1800ccatatatat ga
18127797DNAMentha piperitapromoter(1)..(797) 7aaaaaatggg tacttttatt
cggtaaaatt aattatatta aagatggaca cttcattggc 60tgtggcatcg agcgcgagct
ccatgccgtg tgagcggcac gaccacgact gcctccctca 120tgttttgccc atgcatcgtg
tgttggcacg tcggggcttc ccgtcggtcg tgcaacgggt 180gcgagcctcc gtcgggagag
gtgtctggcg ggccgtacga ttgcacaacc aagactaccc 240tcgagtcgtg gtcgtgcaat
gccacgtcgg gcgccgtgtt cactgtgccg tgcgaaatac 300aagaccaata ttacacgaca
atgtagttgt gtaatcgtgg tcatgtaatt aacttaagag 360gttaatttat cattttacac
gaaaaaataa ctaaaatttg catattgttt tatatgtggc 420taaaatttag ctttacatta
tcaaaatgac tattttgggg attgccctct atatatattg 480tttctaatag tacattttaa
gtgatttcag ataataaata aatacagcca aacaaattaa 540acaacaacgg ctatatagcg
ttgataaatg caccttctag ctagaatgac ttatatagta 600gcataatacg aaatccaaaa
gaaggttgaa gttgtgatgt atttcattaa ttaggccgaa 660aaaatagaaa cgtgcatgct
taatctatag gagtctgtat aaatagaaat atgcatctca 720aattttagaa accagtacgt
cctagaaaaa catagaaaga gagtggaaga aaaggagaaa 780gcaaagatta attaatc
79783514DNAMentha
piperitamodified_base(1)..(3514)The MPTPS4 (1686 bp) gene was cloned in
pHANNIBALvector system by replacing the PDK intron to yield
phannibal-MPTPS4,under tissue specific promoter (Limonene synthase,
797 bp). 8ggccgctcga cgaattaatt ccaatcccac aaaaatctga gcttaacagc
acagttgctc 60ctctcagagc agaatcgggt attcaacacc ctcatatcaa ctactacgtt
gtgtataacg 120gtccacatgc cggtatatac gatgactggg gttgtacaaa ggcggcaaca
aacggcgttc 180ccggagttgc acacaagaaa tttgccacta ttacagaggc aagagcagca
gctgacgcgt 240aaaaaatggg tacttttatt cggtaaaatt aattatatta aagatggaca
cttcattggc 300tgtggcatcg agcgcgagct ccatgccgtg tgagcggcac gaccacgact
gcctccctca 360tgttttgccc atgcatcgtg tgttggcacg tcggggcttc ccgtcggtcg
tgcaacgggt 420gcgagcctcc gtcgggagag gtgtctggcg ggccgtacga ttgcacaacc
aagactaccc 480tcgagtcgtg gtcgtgcaat gccacgtcgg gcgccgtgtt cactgtgccg
tgcgaaatac 540aagaccaata ttacacgaca atgtagttgt gtaatcgtgg tcatgtaatt
aacttaagag 600gttaatttat cattttacac gaaaaaataa ctaaaatttg catattgttt
tatatgtggc 660taaaatttag ctttacatta tcaaaatgac tattttgggg attgccctct
atatatattg 720tttctaatag tacattttaa gtgatttcag ataataaata aatacagcca
aacaaattaa 780acaacaacgg ctatatagcg ttgataaatg caccttctag ctagaatgac
ttatatagta 840gcataatacg aaatccaaaa gaaggttgaa gttgtgatgt atttcattaa
ttaggccgaa 900aaaatagaaa cgtgcatgct taatctatag gagtctgtat aaatagaaat
atgcatctca 960aattttagaa accagtacgt cctagaaaaa catagaaaga gagtggaaga
aaaggagaaa 1020gcaaagatta attaatcgaa ttcatggaaa tttattcccc agttgttcct
gcaatcaaag 1080atgtgaagag gttggatgaa atccgaaaat ccgccaaata ccatccaact
atttggggag 1140acttttttct agcgtataat tccgataata cgaaaatctc tgatggtgaa
gaagaagaag 1200ttgcaaagca gaaagaaatg gtacggaaac tgctagctga agccccagaa
aattccacct 1260acaaaatgga actcatcgat acaatccagc gcctgggagt ggaatatcat
tttgaaaaag 1320aaatcgaaat atccttgaaa tatattcatg agaattatgt gcaccaaaac
agcaaagacg 1380acgatcttca caccgtggct cttcgttttc gtttgcttag acaacaaggt
tacaacgtcc 1440catgcgacgt tttcagcaaa ttcaccgacc gtgaaggaaa ttttgcggcg
gcgctgagaa 1500atgacgttga aagtttgttg gaattatatg aggcgtcgca tctcgcgaca
cgtggcgagg 1560aaattctgga cagcgcaatg gagttgtgtt cttcccatct ccaagcatta
gtaaatgatc 1620agcagttggt gaacaatgtt tctctctcta aacgagtgat tgaagctctg
aagatgccaa 1680ttcgcaagag tctcacgaga ttgggtgcga gaaagttcat ctctctatac
caagaggatg 1740attcgcataa tgaaatactt ttgaattttg cgaaattaga tttcaatata
gtgcagaaga 1800tgcaccagag agagcttagt gatgctacga ggtggtggaa gaagttggac
gtggcgaata 1860aaatgcctta cgcaagagac agaaatgtgg agtgcttctt tttggatggt
gggcgtctac 1920ttcgagcatg ctacgctact gcaagaaaaa tattacttaa atgcataagt
atggcttcaa 1980ttattgatga cacctacgaa tatgcaaccc tacatgaact gcaaattctc
accgacgcta 2040tccaacgttg ggatgttaat gaggcattgg aggattcgcc accatatata
caaatgtgct 2100acagaagcct tattcaaact tatgttgaaa tagaagatga agtagtggag
aaatttggag 2160gagaatcgtc ataccgtgtc caatatgcaa tacaagatat gaaaaaatcg
gtgtgggcat 2220atatggaaga ggcgaaatgg atgtatgacg attatattcc aaccgtggag
gagtatatga 2280aggtttcgat cgtatcttgt ggttatatga caatgtcaac cacttcttta
gtgggtatgg 2340ggatcgatca agtttgcaaa gaagatttcg attggatcgt aaatgaacct
ctcttggttc 2400gagcttcatc ggcaatttgt cgattaaccg acgatctagt cggggacgag
tttgagcaaa 2460aaacgtcgtt ggtggattgt tacatgaaac aacacggtat gtcgaaggac
gaagctcgag 2520ctcatcttcg acaacagttg aaggatgcat ggaaggatat gaaccaagaa
tcccttgagc 2580ctagaccggc ctcgatgcca atcctaacgc gtgttatcaa tctcggccga
gtcatagatc 2640ttctctattc ggaccatgat tgctatagtg atcccaacaa atcgaaaggt
tgggtaaaga 2700tggtgctcgt cgaccccatc gcaatttgag gatcctctag agtcctgctt
taatgagata 2760tgcgagacgc ctatgatcgc atgatatttg ctttcaattc tgttgtgcac
gttgtaaaaa 2820acctgagcat gtgtagctca gatccttacc gccggtttcg gttcattcta
atgaatatat 2880cacccgttac tatcgtattt ttatgaataa tattctccgt tcaatttact
gattgtaccc 2940tactacttat atgtacaata ttaaaatgaa aacaatatat tgtgctgaat
aggtttatag 3000cgacatctat gatagagcgc cacaataaca aacaattgcg ttttattatt
acaaatccaa 3060ttttaaaaaa agcggcagaa ccggtcaaac ctaaaagact gattacataa
atcttattca 3120aatttcaaaa ggccccaggg gctagtatct acgacacacc gagcggcgaa
ctaataacgt 3180tcactgaagg gaactccggt tccccgccgg cgcgcatggg tgagattcct
tgaagttgag 3240tattggccgt ccgctctacc gaaagttacg ggcaccattc aacccggtcc
agcacggcgg 3300ccgggtaacc gacttgctgc cccgagaatt atgcagcatt tttttggtgt
atgtgggccc 3360caaatgaagt gcaggtcaaa ccttgacagt gacgacaaat cgttgggcgg
gtccagggcg 3420aattttgcga caacatgtcg aggctcagca ggacctgcag gcatgcaagc
tagcttacta 3480gtgatgcata ttctatagtg tcacctaaat ctgc
3514915181DNAMentha piperitamodified_base(1)..(15181)The whole
cassette of SEQUENCE ID NO.8 was cloned into pART27 binary vector
system. 9tcgacatctt gctgcgttcg gatattttcg tggagttccc gccacagacc
cggattgaag 60gcgagatcca gcaactcgcg ccagatcatc ctgtgacgga actttggcgc
gtgatgactg 120gccaggacgt cggccgaaag agcgacaagc agatcacgat tttcgacagc
gtcggatttg 180cgatcgagga tttttcggcg ctgcgctacg tccgcgaccg cgttgaggga
tcaagccaca 240gcagcccact cgaccttcta gccgacccag acgagccaag ggatcttttt
ggaatgctgc 300tccgtcgtca ggctttccga cgtttgggtg gttgaacaga agtcattatc
gtacggaatg 360ccagcactcc cgaggggaac cctgtggttg gcatgcacat acaaatggac
gaacggataa 420accttttcac gcccttttaa atatccgtta ttctaataaa cgctcttttc
tcttaggttt 480acccgccaat atatcctgtc aaacactgat agtttaaact gaaggcggga
aacgacaatc 540tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga
cgcgggacaa 600gccgttttac gtttggaact gacagaaccg caacgattga aggagccact
cagccccaat 660acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
acgacaggtt 720tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
tcactcatta 780ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
ttgtgagcgg 840ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
ttaggtgaca 900ctatagaata ctcaagctat gcatccaacg cgttgggagc tctcccatat
cgacctgcag 960gcggccgctc gacgaattaa ttccaatccc acaaaaatct gagcttaaca
gcacagttgc 1020tcctctcaga gcagaatcgg gtattcaaca ccctcatatc aactactacg
ttgtgtataa 1080cggtccacat gccggtatat acgatgactg gggttgtaca aaggcggcaa
caaacggcgt 1140tcccggagtt gcacacaaga aatttgccac tattacagag gcaagagcag
cagctgacgc 1200gtaaaaaatg ggtactttta ttcggtaaaa ttaattatat taaagatgga
cacttcattg 1260gctgtggcat cgagcgcgag ctccatgccg tgtgagcggc acgaccacga
ctgcctccct 1320catgttttgc ccatgcatcg tgtgttggca cgtcggggct tcccgtcggt
cgtgcaacgg 1380gtgcgagcct ccgtcgggag aggtgtctgg cgggccgtac gattgcacaa
ccaagactac 1440cctcgagtcg tggtcgtgca atgccacgtc gggcgccgtg ttcactgtgc
cgtgcgaaat 1500acaagaccaa tattacacga caatgtagtt gtgtaatcgt ggtcatgtaa
ttaacttaag 1560aggttaattt atcattttac acgaaaaaat aactaaaatt tgcatattgt
tttatatgtg 1620gctaaaattt agctttacat tatcaaaatg actattttgg ggattgccct
ctatatatat 1680tgtttctaat agtacatttt aagtgatttc agataataaa taaatacagc
caaacaaatt 1740aaacaacaac ggctatatag cgttgataaa tgcaccttct agctagaatg
acttatatag 1800tagcataata cgaaatccaa aagaaggttg aagttgtgat gtatttcatt
aattaggccg 1860aaaaaataga aacgtgcatg cttaatctat aggagtctgt ataaatagaa
atatgcatct 1920caaattttag aaaccagtac gtcctagaaa aacatagaaa gagagtggaa
gaaaaggaga 1980aagcaaagat taattaatcg aattcatgga aatttattcc ccagttgttc
ctgcaatcaa 2040agatgtgaag aggttggatg aaatccgaaa atccgccaaa taccatccaa
ctatttgggg 2100agactttttt ctagcgtata attccgataa tacgaaaatc tctgatggtg
aagaagaaga 2160agttgcaaag cagaaagaaa tggtacggaa actgctagct gaagccccag
aaaattccac 2220ctacaaaatg gaactcatcg atacaatcca gcgcctggga gtggaatatc
attttgaaaa 2280agaaatcgaa atatccttga aatatattca tgagaattat gtgcaccaaa
acagcaaaga 2340cgacgatctt cacaccgtgg ctcttcgttt tcgtttgctt agacaacaag
gttacaacgt 2400cccatgcgac gttttcagca aattcaccga ccgtgaagga aattttgcgg
cggcgctgag 2460aaatgacgtt gaaagtttgt tggaattata tgaggcgtcg catctcgcga
cacgtggcga 2520ggaaattctg gacagcgcaa tggagttgtg ttcttcccat ctccaagcat
tagtaaatga 2580tcagcagttg gtgaacaatg tttctctctc taaacgagtg attgaagctc
tgaagatgcc 2640aattcgcaag agtctcacga gattgggtgc gagaaagttc atctctctat
accaagagga 2700tgattcgcat aatgaaatac ttttgaattt tgcgaaatta gatttcaata
tagtgcagaa 2760gatgcaccag agagagctta gtgatgctac gaggtggtgg aagaagttgg
acgtggcgaa 2820taaaatgcct tacgcaagag acagaaatgt ggagtgcttc tttttggatg
gtgggcgtct 2880acttcgagca tgctacgcta ctgcaagaaa aatattactt aaatgcataa
gtatggcttc 2940aattattgat gacacctacg aatatgcaac cctacatgaa ctgcaaattc
tcaccgacgc 3000tatccaacgt tgggatgtta atgaggcatt ggaggattcg ccaccatata
tacaaatgtg 3060ctacagaagc cttattcaaa cttatgttga aatagaagat gaagtagtgg
agaaatttgg 3120aggagaatcg tcataccgtg tccaatatgc aatacaagat atgaaaaaat
cggtgtgggc 3180atatatggaa gaggcgaaat ggatgtatga cgattatatt ccaaccgtgg
aggagtatat 3240gaaggtttcg atcgtatctt gtggttatat gacaatgtca accacttctt
tagtgggtat 3300ggggatcgat caagtttgca aagaagattt cgattggatc gtaaatgaac
ctctcttggt 3360tcgagcttca tcggcaattt gtcgattaac cgacgatcta gtcggggacg
agtttgagca 3420aaaaacgtcg ttggtggatt gttacatgaa acaacacggt atgtcgaagg
acgaagctcg 3480agctcatctt cgacaacagt tgaaggatgc atggaaggat atgaaccaag
aatcccttga 3540gcctagaccg gcctcgatgc caatcctaac gcgtgttatc aatctcggcc
gagtcataga 3600tcttctctat tcggaccatg attgctatag tgatcccaac aaatcgaaag
gttgggtaaa 3660gatggtgctc gtcgacccca tcgcaatttg aggatcctct agagtcctgc
tttaatgaga 3720tatgcgagac gcctatgatc gcatgatatt tgctttcaat tctgttgtgc
acgttgtaaa 3780aaacctgagc atgtgtagct cagatcctta ccgccggttt cggttcattc
taatgaatat 3840atcacccgtt actatcgtat ttttatgaat aatattctcc gttcaattta
ctgattgtac 3900cctactactt atatgtacaa tattaaaatg aaaacaatat attgtgctga
ataggtttat 3960agcgacatct atgatagagc gccacaataa caaacaattg cgttttatta
ttacaaatcc 4020aattttaaaa aaagcggcag aaccggtcaa acctaaaaga ctgattacat
aaatcttatt 4080caaatttcaa aaggccccag gggctagtat ctacgacaca ccgagcggcg
aactaataac 4140gttcactgaa gggaactccg gttccccgcc ggcgcgcatg ggtgagattc
cttgaagttg 4200agtattggcc gtccgctcta ccgaaagtta cgggcaccat tcaacccggt
ccagcacggc 4260ggccgggtaa ccgacttgct gccccgagaa ttatgcagca tttttttggt
gtatgtgggc 4320cccaaatgaa gtgcaggtca aaccttgaca gtgacgacaa atcgttgggc
gggtccaggg 4380cgaattttgc gacaacatgt cgaggctcag caggacctgc aggcatgcaa
gctagcttac 4440tagtgatgca tattctatag tgtcacctaa atctgcggcc gcactagtga
tatcccgcgg 4500ccatggcggc cgggagcatg cgacgtcggg cccaattcgc cctatagtga
gtcgtattac 4560aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
tacccaactt 4620aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
ggcccgcacc 4680gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
aacgttaatg 4740ggtttctgga gtttaatgag ctaagcacat acgtcagaaa ccattattgc
gcgttcaaaa 4800gtcgcctaag gtcactatca gctagcaaat atttcttgtc aaaaatgctc
cactgacgtt 4860ccataaattc ccctcggtat ccaattagag tctcatattc actctcaatc
caaataatct 4920gcaatggcaa ttaccttatc cgcaacttct ttacctattt ccgcccggat
ccgggcaggt 4980tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
gacaatcggc 5040tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
ttttgtcaag 5100accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
atcgtggctg 5160gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
gggaagggac 5220tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
tgctcctgcc 5280gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
tccggctacc 5340tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
gatggaagcc 5400ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
agccgaactg 5460ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
ccatggcgat 5520gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
cgactgtggc 5580cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
tattgctgaa 5640gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
cgctcccgat 5700tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
actctggggt 5760tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
tccaccgccg 5820ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
atgatcctcc 5880agcgcgggga tctcatgctg gagttcttcg cccaccccga tccaacactt
acgtttgcaa 5940cgtccaagag caaatagacc acgaacgccg gaaggttgcc gcagcgtgtg
gattgcgtct 6000caattctctc ttgcaggaat gcaatgatga atatgatact gactatgaaa
ctttgaggga 6060atactgccta gcaccgtcac ctcataacgt gcatcatgca tgccctgaca
acatggaaca 6120tcgctatttt tctgaagaat tatgctcgtt ggaggatgtc gcggcaattg
cagctattgc 6180caacatcgaa ctacccctca cgcatgcatt catcaatatt attcatgcgg
ggaaaggcaa 6240gattaatcca actggcaaat catccagcgt gattggtaac ttcagttcca
gcgacttgat 6300tcgttttggt gctacccacg ttttcaataa ggacgagatg gtggagtaaa
gaaggagtgc 6360gtcgaagcag atcgttcaaa catttggcaa taaagtttct taagattgaa
tcctgttgcc 6420ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt
aataattaac 6480atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc
gcaattatac 6540atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt
atcgcgcgcg 6600gtgtcatcta tgttactaga tcgaattaat tccaggcggt gaagggcaat
cagctgttgc 6660ccgtctcact ggtgaaaaga aaaaccaccc cagtacatta aaaacgtccg
caatgtgtta 6720ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgccacc
agccagccaa 6780cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag
cccatcagtc 6840cgggacggcg tcagcgggag agccgttgta aggcggcaga ctttgctcat
gttaccgatg 6900ctattcggaa gaacggcaac taagctgccg ggtttgaaac acggatgatc
tcgcggaggg 6960tagcatgttg attgtaacga tgacagagcg ttgctgcctg tgatcaaata
tcatctccct 7020cgcagagatc cgaattatca gccttcttat tcatttctcg cttaaccgtg
acaggctgtc 7080gatcttgaga actatgccga cataatagga aatcgctgga taaagccgct
gaggaagctg 7140agtggcgcta tttctttaga agtgaacgtt gacgatgtcg acggatcttt
tccgctgcat 7200aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt
ttcgcacgat 7260atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa
cggcgtcagc 7320cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc
actgtccctt 7380attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
gctaccgccg 7440gcgtaacaga tgagggcaag cggatggctg atgaaaccaa gccaaccagg
ggtgatgctg 7500ccaacttact gatttagtgt atgatggtgt ttttgaggtg ctccagtggc
ttctgtttct 7560atcagctgtc cctcctgttc agctactgac ggggtggtgc gtaacggcaa
aagcaccgcc 7620ggacatcagc gctatctctg ctctcactgc cgtaaaacat ggcaactgca
gttcacttac 7680accgcttctc aacccggtac gcaccagaaa atcattgata tggccatgaa
tggcgttgga 7740tgccgggcaa cagcccgcat tatgggcgtt ggcctcaaca cgattttacg
tcacttaaaa 7800aactcaggcc gcagtcggta acctcgcgca tacagccggg cagtgacgtc
atcgtctgcg 7860cggaaatgga cgaacagtgg ggctatgtcg gggctaaatc gcgccagcgc
tggctgtttt 7920acgcgtatga cagtctccgg aagacggttg ttgcgcacgt attcggtgaa
cgcactatgg 7980cgacgctggg gcgtcttatg agcctgctgt caccctttga cgtggtgata
tggatgacgg 8040atggctggcc gctgtatgaa tcccgcctga agggaaagct gcacgtaatc
agcaagcgat 8100atacgcagcg aattgagcgg cataacctga atctgaggca gcacctggca
cggctgggac 8160ggaagtcgct gtcgttctca aaatcggtgg agctgcatga caaagtcatc
gggcattatc 8220tgaacataaa acactatcaa taagttggag tcattaccca accaggaagg
gcagcccacc 8280tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg
cggcggcggc 8340cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa
tcacgggcgt 8400cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg
gccgcctggg 8460cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg
gtgatgccac 8520gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca
aggtcatgat 8580gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac
ggccgggggg 8640tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac
ttcgcggagc 8700tggtattcgt gcagggcaag attcggaata ccaagtacga gaaggacggc
cagacggtct 8760acgggaccga cttcattgcc gataaggtgg attatctgga caccaaggca
ccaggcgggt 8820caaatcagga ataagggcac attgccccgg cgtgagtcgg ggcaatcccg
caaggagggt 8880gaatgaatcg gacgtttgac cggaaggcat acaggcaaga actgatcgac
gcggggtttt 8940ccgccgagga tgccgaaacc atcgcaagcc gcaccgtcat gcgtgcgccc
cgcgaaacct 9000tccagtccgt cggctcgatg gtccagcaag ctacggccaa gatcgagcgc
gacagcgtgc 9060aactggctcc ccctgccctg cccgcgccat cggccgccgt ggagcgttcg
cgtcgtctcg 9120aacaggaggc ggcaggtttg gcgaagtcga tgaccatcga cacgcgagga
actatgacga 9180ccaagaagcg aaaaaccgcc ggcgaggacc tggcaaaaca ggtcagcgag
gccaagcagg 9240ccgcgttgct gaaacacacg aagcagcaga tcaaggaaat gcagctttcc
ttgttcgata 9300ttgcgccgtg gccggacacg atgcgagcga tgccaaacga cacggcccgc
tctgccctgt 9360tcaccacgcg caacaagaaa atcccgcgcg aggcgctgca aaacaaggtc
attttccacg 9420tcaacaagga cgtgaagatc acctacaccg gcgtcgagct gcgggccgac
gatgacgaac 9480tggtgtggca gcaggtgttg gagtacgcga agcgcacccc tatcggcgag
ccgatcacct 9540tcacgttcta cgagctttgc caggacctgg gctggtcgat caatggccgg
tattacacga 9600aggccgagga atgcctgtcg cgcctacagg cgacggcgat gggcttcacg
tccgaccgcg 9660ttgggcacct ggaatcggtg tcgctgctgc accgcttccg cgtcctggac
cgtggcaaga 9720aaacgtcccg ttgccaggtc ctgatcgacg aggaaatcgt cgtgctgttt
gctggcgacc 9780actacacgaa attcatatgg gagaagtacc gcaagctgtc gccgacggcc
cgacggatgt 9840tcgactattt cagctcgcac cgggagccgt acccgctcaa gctggaaacc
ttccgcctca 9900tgtgcggatc ggattccacc cgcgtgaaga agtggcgcga gcaggtcggc
gaagcctgcg 9960aagagttgcg aggcagcggc ctggtggaac acgcctgggt caatgatgac
ctggtgcatt 10020gcaaacgcta gggccttgtg gggtcagttc cggctggggg ttcagcagcc
agcgctttac 10080tggcatttca ggaacaagcg ggcactgctc gacgcacttg cttcgctcag
tatcgctcgg 10140gacgcacggc gcgctctacg aactgccgat aaacagagga ttaaaattga
caattgtgat 10200taaggctcag attcgacggc ttggagcggc cgacgtgcag gatttccgcg
agatccgatt 10260gtcggccctg aagaaagctc cagagatgtt cgggtccgtt tacgagcacg
aggagaaaaa 10320gcccatggag gcgttcgctg aacggttgcg agatgccgtg gcattcggcg
cctacatcga 10380cggcgagatc attgggctgt cggtcttcaa acaggaggac ggccccaagg
acgctcacaa 10440ggcgcatctg tccggcgttt tcgtggagcc cgaacagcga ggccgagggg
tcgccggtat 10500gctgctgcgg gcgttgccgg cgggtttatt gctcgtgatg atcgtccgac
agattccaac 10560gggaatctgg tggatgcgca tcttcatcct cggcgcactt aatatttcgc
tattctggag 10620cttgttgttt atttcggtct accgcctgcc gggcggggtc gcggcgacgg
taggcgctgt 10680gcagccgctg atggtcgtgt tcatctctgc cgctctgcta ggtagcccga
tacgattgat 10740ggcggtcctg ggggctattt gcggaactgc gggcgtggcg ctgttggtgt
tgacaccaaa 10800cgcagcgcta gatcctgtcg gcgtcgcagc gggcctggcg ggggcggttt
ccatggcgtt 10860cggaaccgtg ctgacccgca agtggcaacc tcccgtgcct ctgctcacct
ttaccgcctg 10920gcaactggcg gccggaggac ttctgctcgt tccagtagct ttagtgtttg
atccgccaat 10980cccgatgcct acaggaacca atgttctcgg cctggcgtgg ctcggcctga
tcggagcggg 11040tttaacctac ttcctttggt tccgggggat ctcgcgactc gaacctacag
ttgtttcctt 11100actgggcttt ctcagccggg atggcgctaa gaagctattg ccgccgatct
tcatatgcgg 11160tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
ttccgcttcc 11220tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
agctcactca 11280aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
catgtgagca 11340aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg 11400ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg 11460acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt 11520ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
cgtggcgctt 11580tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc 11640tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt 11700gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
taacaggatt 11760agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
taactacggc 11820tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
cttcggaaaa 11880agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt 11940tgcaagcagc agattacgcg cagaaaaaaa ggatatcaag aagatccttt
gatcttttct 12000acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
catgagatta 12060tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
atcaatctaa 12120agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
ggcacctatc 12180tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact 12240acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
agacccacgc 12300tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
gcgcagaagt 12360ggtcctgcaa ctttatccgc ctccatccag tctattaaac aagtggcagc
aacggattcg 12420caaacctgtc acgccttttg tgccaaaagc cgcgccaggt ttgcgatccg
ctgtgccagg 12480cgttaggcgt catatgaaga tttcggtgat ccctgagcag gtggcggaaa
cattggatgc 12540tgagaaccat ttcattgttc gtgaagtgtt cgatgtgcac ctatccgacc
aaggctttga 12600actatctacc agaagtgtga gcccctaccg gaaggattac atctcggatg
atgactctga 12660tgaagactct gcttgctatg gcgcattcat cgaccaagag cttgtcggga
agattgaact 12720caactcaaca tggaacgatc tagcctctat cgaacacatt gttgtgtcgc
acacgcaccg 12780aggcaaagga gtcgcgcaca gtctcatcga atttgcgaaa aagtgggcac
taagcagaca 12840gctccttggc atacgattag agacacaaac gaacaatgta cctgcctgca
atttgtacgc 12900aaaatgtggc tttactctcg gcggcattga cctgttcacg tataaaacta
gacctcaagt 12960ctcgaacgaa acagcgatgt actggtactg gttctcggga gcacaggatg
acgcctaaca 13020attcattcaa gccgacaccg cttcgcggcg cggcttaatt caggagttaa
acatcatgag 13080ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
tcatcgagcg 13140ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
atggcggcct 13200gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
atgaaacaac 13260gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
agagcgagat 13320tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
ggcgttatcc 13380agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
caggtatctt 13440cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
gagaacatag 13500cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
aacaggatct 13560atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
gggctggcga 13620tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
ccggcaaaat 13680cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
agtatcagcc 13740cgtcatactt gaagctaggc aggcttatct tggacaagaa gatcgcttgg
cctcgcgcgc 13800agatcagttg gaagaatttg ttcactacgt gaaaggcgag atcaccaagg
tagtcggcaa 13860ataatgtcta acaattcgtt caagccgacg ccgcttcgcg gcgcggctta
actcaagcgt 13920tagagagctg gggaagacta tgcgcgatct gttgaaggtg gttctaagcc
tcgtacttgc 13980gatggcatcg gggcaggcac ttgctgacct gccaattgtt ttagtggatg
aagctcgtct 14040tccctatgac tactccccat ccaactacga catttctcca agcaactacg
acaactccat 14100aagcaattac gacaatagtc catcaaatta cgacaactct gagagcaact
acgataatag 14160ttcatccaat tacgacaata gtcgcaacgg aaatcgtagg cttatatata
gcgcaaatgg 14220gtctcgcact ttcgccggct actacgtcat tgccaacaat gggacaacga
acttcttttc 14280cacatctggc aaaaggatgt tctacacccc aaaagggggg cgcggcgtct
atggcggcaa 14340agatgggagc ttctgcgggg cattggtcgt cataaatggc caattttcgc
ttgccctgac 14400agataacggc ctgaagatca tgtatctaag caactagcct gctctctaat
aaaatgttag 14460gagcttggct gccatttttg gggtgaggcc gttcgcggcc gaggggcgca
gcccctgggg 14520ggatgggagg cccgcgttag cgggccggga gggttcgaga agggggggca
ccccccttcg 14580gcgtgcgcgg tcacgcgcca gggcgcagcc ctggttaaaa acaaggttta
taaatattgg 14640tttaaaagca ggttaaaaga caggttagcg gtggccgaaa aacgggcgga
aacccttgca 14700aatgctggat tttctgcctg tggacagccc ctcaaatgtc aataggtgcg
cccctcatct 14760gtcagcactc tgcccctcaa gtgtcaagga tcgcgcccct catctgtcag
tagtcgcgcc 14820cctcaagtgt caataccgca gggcacttat ccccaggctt gtccacatca
tctgtgggaa 14880actcgcgtaa aatcaggcgt tttcgccgat ttgcgaggct ggccagctcc
acgtcgccgg 14940ccgaaatcga gcctgcccct catctgtcaa cgccgcgccg ggtgagtcgg
cccctcaagt 15000gtcaacgtcc gcccctcatc tgtcagtgag ggccaagttt tccgcgaggt
atccacaacg 15060ccggcggccg gccgcggtgt ctcgcacacg gcttcgacgg cgtttctggc
gcgtttgcag 15120ggccatagac ggccgccagc ccagcggcga gggcaaccag cccggtgagc
gtcggaaagg 15180g
1518110707DNAMentha piperitaterminator(1)..(707) 10ctgctttaat
gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgtt 60gtgcacgttg
taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttc 120attctaatga
atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaa 180tttactgatt
gtaccctact acttatatgt acaatattaa aatgaaaaca atatattgtg 240ctgaataggt
ttatagcgac atctatgata gagcgccaca ataacaaaca attgcgtttt 300attattacaa
atccaatttt aaaaaaagcg gcagaaccgg tcaaacctaa aagactgatt 360acataaatct
tattcaaatt tcaaaaggcc ccaggggcta gtatctacga cacaccgagc 420ggcgaactaa
taacgttcac tgaagggaac tccggttccc cgccggcgcg catgggtgag 480attccttgaa
gttgagtatt ggccgtccgc tctaccgaaa gttacgggca ccattcaacc 540cggtccagca
cggcggccgg gtaaccgact tgctgccccg agaattatgc agcatttttt 600tggtgtatgt
gggccccaaa tgaagtgcag gtcaaacctt gacagtgacg acaaatcgtt 660gggcgggtcc
agggcgaatt ttgcgacaac atgtcgaggc tcagcag
7071130DNAMentha piperitaprimer_bind(1)..(30)Primers for cloning MPTPS4
gene in Overexpression vector (pHANNIBAL) MPTPS4_F_oe_Eco
11agaattcatg gaaatttatt ccccagttgt
301229DNAMentha piperitaprimer_bind(1)..(29)Primers for cloning MPTPS4
genein Overexpression vector (pHANNIBAL) MPTPS4_R_oe_Bam
12ggatcctcaa attgcgatgg ggtcgacga
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